Isolated human zinc metalloprotease, nucleic acid molecules encoding said enzymes, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of enzyme proteins that arerelated to the metalloprotease enzyme subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins and nucleic acid molecules encodingsuch peptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

[0002] Many human enzymes serve as targets for the action ofpharmaceutically active compounds. Several classes of human enzymes thatserve as such targets include helicase, steroid esterase and sulfatase,convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase,glutanase, decarboxylase, isomerase and reductase. It is theforeimportant in developing new pharmaceutical compounds to identify targetenzyme proteins that can be put into high-throughput screening formats.The present invention advances the state of the art by providing novelhuman drug target enzymes related to the metalloprotease subfamily.

[0003] Endothelin-Converting Enzymes

[0004] The novel human protein, and encoding gene, provided by thepresent invention is related to the family of metalloprotease enzymes(also referred to as the peptidase family M13, zinc metalloproteasefamily, and the neprilysin family) in general and shows a high degree ofsimilarity to the endothelin-converting enzyme subfamily ofmetalloproteases. Furthermore, the protein of the present invention maybe a novel isoform of the gene provided in Genbank gi7662200 (see theamino acid sequence alignment in FIG. 2).

[0005] Endothelin-coverting enzymes (ECE) are membrane-boundmetalloproteases that catalyze the proteolytic activation ofendothelins, which are potent vasoactive peptides. Endothelins areproduced from biologically inactive intermediates known as bigendothelins by ECE-catalyzed proteolytic processing. ECE function insecretory pathways as well as on the cell surface. ECE-1 and ECE-2 havebeen characterized. ECE-2 is structurally related to ECE-1, neuralendopeptidase 24.11, and human Kell blood group protein. ECE-1 and ECE-2are both inhibited by phosphoramidon. ECE-1 is most active at neutralpH, whereas an acidic pH is optimum for ECE-2. It is though that ECE-2converts endogenously synthesized big endothelin-1 to matureendothelin-1 at the acidic environement of the trans-Golgi network(Emoto et al., J. Biol Chem 1995 June 23;270(25):15262-8).

[0006] Metalloproteases

[0007] The metalloproteases may be one of the older classes ofproteinases and are found in bacteria, fungi as well as in higherorganisms. They differ widely in their sequences and their structuresbut the great majority of enzymes contain a zinc atom which iscatalytically active. In some cases, zinc may be replaced by anothermetal such as cobalt or nickel without loss of the activity. Bacterialthermolysin has been well characterized and its crystallographicstructure indicates that zinc is bound by two histidines and oneglutamic acid. Many enzymes contain the sequence HEXXH, which providestwo histidine ligands for the zinc whereas the third ligand is either aglutamic acid (thermolysin, neprilysin, alanyl aminopeptidase) or ahistidine (astacin). Other families exhibit a distinct mode of bindingof the Zn atom. The catalytic mechanism leads to the formation of a noncovalent tetrahedral intermediate after the attack of a zinc-bound watermolecule on the carbonyl group of the scissile bond. This intermediateis further decomposed by transfer of the glutamic acid proton to theleaving group.

[0008] Metalloproteases contain a catalytic zinc metal center whichparticipates in the hydrolysis of the peptide backbone (reviewed inPower and Harper, in Protease Inhibitors, A. J. Barrett and G. Salversen(eds.) Elsevier, Amsterdam, 1986, p. 219). The active zinc centerdifferentiates some of these proteases from calpains and trypsins whoseactivities are dependent upon the presence of calcium. Examples ofmetalloproteases include carboxypeptidase A, carboxypeptidase B, andthermolysin.

[0009] Metalloproteases have been isolated from a number of procaryoticand eucaryotic sources, e.g. Bacillus subtilis (McConn et al., 1964, J.Biol. Chem. 239:3706); Bacillus megaterium; Serratia (Miyata et al.,1971, Agr. Biol. Chem. 35:460); Clostridium bifennentans (MacFarlane etal., 1992, App. Environ. Microbiol. 58:1195-1200), Legionellapneumophila (Moffat et al., 1994, Infection and Immunity 62:751-3). Inparticular, acidic metalloproteases have been isolated from broad-bandedcopperhead venoms (Johnson and Ownby, 1993, Int. J. Biochem.25:267-278), rattlesnake venoms (Chlou et al., 1992, Biochem. Biophys.Res. Commun. 187:389-396) and articular cartilage (Treadwell et al.,1986, Arch. Biochem. Biophys. 251:715-723). Neutral metalloproteases,specifically those having optimal activity at neutral pH have, forexample, been isolated from Aspergillus sojae (Sekine, 1973, Agric.Biol. Chem. 37:1945-1952). Neutral metalloproteases obtained fromAspergillus have been classified into two groups, npI and npII (Sekine,1972, Agric. Biol. Chem. 36:207-216). So far, success in obtaining aminoacid sequence information from these fungal neutral metalloproteases hasbeen limited. An npII metalloprotease isolated from Aspergillus oryzaehas been cloned based on amino acid sequence presented in the literature(Tatsumi et al., 1991, Mol. Gen. Genet. 228:97-103). However, to date,no npI fungal metalloprotease has been cloned or sequenced. Alkalinemetalloproteases, for example, have been isolated from Pseudomonasacruginosa (Baumann et al., 1993, EMBO J. 12:3357-3364) and the insectpathogen Xenorhabdus luminescens (Schmidt et al., 1998, Appl. Environ.Microbiol. 54:2793-2797).

[0010] Metalloproteases have been devided into several distinct familiesbased primarily on activity and sturcture: 1) water nucleophile; waterbound by single zinc ion ligated to two His (within the motif HEXXH) andGlu, His or Asp; 2) water nucleophile; water bound by single zinc ionligated to His, Glu (within the motif HXXE) and His; 3) waternucleophile; water bound by single zinc ion ligated to His, Asp and His;4) Water nucleophile; water bound by single zinc ion ligated to two His(within the motif HXXEH) and Glu and 5) water nucleophile; water boundby two zinc ions ligated by Lys, Asp, Asp, Asp, Glu.

[0011] Examples of members of the metalloproteinase family include, butare not limited to, membrane alanyl aminopeptidase (Homo sapiens),germinal peptidyl-dipeptidase A (Homo sapiens), thimet oligopeptidase(Rattus norvegicus), oligopeptidase F (Lactococcus lactis), mycolysin(Streptomyces cacaoi), immune inhibitor A (Bacillus thuringiensis),snapalysin (Streptomyces lividans), leishmanolysin (Leishmania major),microbial collagenase (Vibrio alginolyticus), microbial collagenase,class I (Clostridium perfringens), collagenase 1 (Homo sapiens),serralysin (Serratia marcescens), fragilysin (Bacteroides fragilis),gametolysin (Chlamydomonas reinhardtii), astacin (Astacus fluviatilis),adamalysin (Crotalus adamanteus), ADAM 10 (Bos taurus), neprilysin (Homosapiens), carboxypeptidase A (Homo sapiens), carboxypeptidase E (Bostaurus), gamma-D-glutamyl-(L)-meso-diaminopimelate peptidase I (Bacillussphaericus), vanY D-Ala-D-Ala carboxypeptidase (Enterococcus faecium),endolysin (bacteriophage A118), pitrilysin (Escherichia coli),mitochondrial processing peptidase (Saccharomyces cerevisiae), leucylaminopeptidase (Bos taurus), aminopeptidase I (Saccharomycescerevisiae), membrane dipeptidase (Homo sapiens), glutamatecarboxypeptidase (Pseudomonas sp.), Gly-X carboxypeptidase(Saccharomyces cerevisiae), O-sialoglycoprotein endopeptidase(Pasteurella haemolytica), beta-lytic metalloendopeptidase(Achromobacter lyticus), methionyl aminopeptidase I (Escherichia coli),X-Pro aminopeptidase (Escherichia coli), X-His dipeptidase (Escherichiacoli), IgA1-specific metalloendopeptidase (Streptococcus sanguis),tentoxilysin (Clostridium tetani), leucyl aminopeptidase (Vibrioproteolyticus), aminopeptidase (Streptomyces griseus), LAPaminopeptidase (Escherichia coli), aminopeptidase T (Thermus aquaticus),hyicolysin (Staphylococcus hyicus), carboxypeptidase Taq (Thermusaquaticus), anthrax lethal factor (Bacillus anthracis), penicillolysin(Penicillium citrinum), fungalysin (Aspergillus fumigatus), lysostaphin(Staphylococcus simulans), beta-aspartyl dipeptidase (Escherichia coli),carboxypeptidase Ss1 (Sulfolobus solfataricus), FtsH endopeptidase(Escherichia coli), glutamyl aminopeptidase (Lactococcus lactis),cytophagalysin (Cytophaga sp.), metalloendopeptidase (vaccinia virus),VanX D-Ala-D-Ala dipeptidase (Enterococcus faecium), Ste24pendopeptidase (Saccharomyces cerevisiae), dipeptidyl-peptidase III(Rattus norvegicus), S2P protease (Homo sapiens), sporulation factorSpoIVFB (Bacillus subtilis), and HYBD endopeptidase (Escherichia coli).

[0012] Metalloproteases have been found to have a number of uses. Forexample, there is strong evidence that a metalloprotease is involved inthe in vivo proteolytic processing of the vasoconstrictor, endothelin-1.Rat metalloprotease has been found to be involved in peptide hormoneprocessing. One important subfamily of the metalloproteases are thematrix metalloproteases.

[0013] A number of diseases are thought to be mediated by excess orundesired metalloprotease activity or by an imbalance in the ratio ofthe various members of the protease family of proteins. These include:a) osteoarthritis (Woessner, et al., J. Biol.Chem. 259(6), 3633, 1984;Phadke, et al., J. Rheumatol. 10, 852, 1983), b) rheumatoid arthritis(Mullins, et al., Biochim. Biophys. Acta 695, 117, 1983; Woolley, etal., Arthritis Rheum. 20, 1231, 1977; Gravallese, et al., ArthritisRheum. 34, 1076, 1991), c) septic arthritis (Williams, et al., ArthritisRheum. 33, 533, 1990), d) tumor metastasis (Reich, et al., Cancer Res.48, 3307, 1988, and Matrisian, et al., Proc. Nat'l. Acad. Sci., USA 83,9413, 1986), e) periodontal diseases (Overall, et al., J. PeriodontalRes. 22, 81, 1987), f) corneal ulceration (Burns, et al., Invest.Opthalmol. Vis. Sci. 30, 1569, 1989), g) proteinuria (Baricos, et al.,Biochem. J. 254, 609, 1988), h) coronary thrombosis from atheroscleroticplaque rupture (Henney, et al., Proc. Nat'l. Acad. Sci., USA 88, 8154-8158, 1991), i) aneurysmal aortic disease (Vine, et al., Clin. Sci. 81,233, 1991), j) birth control (Woessner, et al., Steroids 54, 491, 1989),k) dystrophobic epidermolysis bullosa (Kronberger, et al., J. Invest.Dermatol. 79, 208, 1982), and 1) degenerative cartilage loss followingtraumatic joint injury, m) conditions leading to inflammatory responses,osteopenias mediated by MMP activity, n) tempero mandibular jointdisease, o) demyelating diseases of the nervous system (Chantry, et al.,J. Neurochem. 50, 688, 1988).

[0014] Proteases and Cancer

[0015] Proteases are critical elements at several stages in theprogression of metastatic cancer. In this process, the proteolyticdegradation of structural protein in the basal membrane allows forexpansion of a tumor in the primary site, evasion from this site as wellas homing and invasion in distant, secondary sites. Also, tumor inducedangiogenesis is required for tumor growth and is dependent onproteolytic tissue remodeling. Transfection experiments with varioustypes of proteases have shown that the matrix metalloproteases play adominant role in these processes in particular gelatinases A and B(MMP-2 and MMP-9, respectively). For an overview of this field seeMullins, et al., Biochim. Biophys. Acta 695, 177, 1983; Ray, et al.,Eur. Respir. J. 7, 2062, 1994; Birkedal-Hansen, et al., Crit. Rev. OralBiol. Med. 4, 197, 1993.

[0016] Furthermore, it was demonstrated that inhibition of degradationof extracellular matrix by the native matrix metalloprotease inhibitorTIMP-2 (a protein) arrests cancer growth (DeClerck, et al., Cancer Res.52, 701, 1992) and that TIMP-2 inhibits tumor-induced angiogenesis inexperimental systems (Moses, et al. Science 248, 1408, 1990). For areview, see DeClerck, et al., Ann. N. Y. Acad. Sci. 732, 222, 1994. Itwas further demonstrated that the synthetic matrix metalloproteaseinhibitor batimastat when given intraperitoneally inhibits human colontumor growth and spread in an orthotopic model in nude mice (Wang, etal. Cancer Res. 54, 4726, 1994) and prolongs the survival of micebearing human ovarian carcinoma xenografts (Davies, et. al., Cancer Res.53, 2087, 1993). The use of this and related compounds has beendescribed in Brown, et al., WO-9321942 A2.

[0017] There are several patents and patent applications claiming theuse of metalloprotease inhibitors for the retardation of metastaticcancer, promoting tumor regression, inhibiting cancer cellproliferation, slowing or preventing cartilage loss associated withosteoarthritis or for treatment of other diseases as noted above (e.g.Levy, et al., WO-9519965 A1; Beckett, et al., WO-9519956 A1; Beckett, etal., WO-9519957 A1;Beckett, et al., WO-9519961 A1;Brown, et al.,WO-9321942 A2; Crimmin, et al., WO-9421625 A1; Dickens, et al., U.S.Pat. No. 4,599,361; Hughes, et al., U.S. Pat. No. 5,190,937; Broadhurst,et al., EP 574758 A1; Broadhurst, et al., EP 276436; and Myers, et al.,EP 520573 A1.

[0018] Enzyme proteins, particularly members of the metalloproteaseenzyme subfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of enzyme proteins. The present invention advances the stateof the art by providing previously unidentified human enzyme proteins,and the polynucleotides encoding them, that have homology to members ofthe metalloprotease enzyme subfamily. These novel compositions areuseful in the diagnosis, prevention and treatment of biologicalprocesses associated with human diseases.

SUMMARY OF THE INVENTION

[0019] The present invention is based in part on the identification ofamino acid sequences of human enzyme peptides and proteins that arerelated to the metalloprotease enzyme subfamily, as well as allelicvariants and other mammalian orthologs thereof. These unique peptidesequences, and nucleic acid sequences that encode these peptides, can beused as models for the development of human therapeutic targets, aid inthe identification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate enzyme activity incells and tissues that express the enzyme. Experimental data as providedin FIG. 1 indicates expression in humans in the lung, amygdala, adrenalgland, hippocampus, and fetus.

DESCRIPTION OF THE FIGURE SHEETS

[0020]FIG. 1 provides the nucleotide sequence of a cDNA molecule thatencodes the enzyme protein of the present invention. (SEQ ID NO: 1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in the lung, amygdala, adrenal gland, hippocampus, and fetus.

[0021]FIG. 2 provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO: 2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0022]FIG. 3 provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO: 3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 4 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0023] General Description

[0024] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a enzyme protein or part of aenzyme protein and are related to the metalloprotease enzyme subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human enzyme peptides and proteins that are related to themetalloprotease enzyme subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these enzyme peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the enzyme of the present invention.

[0025] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known enzyme proteins of themetalloprotease enzyme subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the lung, amygdala, adrenal gland, hippocampus, and fetus. The arthas clearly established the commercial importance of members of thisfamily of proteins and proteins that have expression patterns similar tothat of the present gene. Some of the more specific features of thepeptides of the present invention, and the uses thereof, are describedherein, particularly in the Background of the Invention and in theannotation provided in the Figures, and/or are known within the art foreach of the known metalloprotease family or subfamily of enzymeproteins.

[0026] Specific Embodiments

[0027] Peptide Molecules

[0028] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the metalloprotease enzymesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the enzyme peptides of the present invention, enzyme peptides,or peptides/proteins of the present invention.

[0029] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the enzyme peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0030] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0031] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0032] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0033] The isolated enzyme peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the lung, amygdala, adrenal gland, hippocampus, and fetus. Forexample, a nucleic acid molecule encoding the enzyme peptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

[0034] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO: 3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0035] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO: 3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0036] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQID NO: 3). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0037] The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

[0038] In some uses, the fusion protein does not affect the activity ofthe enzyme peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0039] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the enzyme peptide.

[0040] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0041] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the enzyme peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0042] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0043] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

[0044] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0045] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the enzyme peptides of the present invention as well as beingencoded by the same genetic locus as the enzyme peptide provided herein.The gene encoding the novel enzyme of the present invention is locatedon a genome component that has been mapped to human chromosome 3 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

[0046] Allelic variants of a enzyme peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel enzyme of the presentinvention is located on a genome component that has been mapped to humanchromosome 3 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data. As used herein, twoproteins (or a region of the proteins) have significant homology whenthe amino acid sequences are typically at least about 70-80%, 80-90%,and more typically at least about 90-95% or more homologous. Asignificantly homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence that willhybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0047]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme of the present invention. SNPs were identifiedat 4 different nucleotide positions. Some of these SNPs that are locatedoutside the ORF and in introns may affect gene transcription.

[0048] Paralogs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0049] Orthologs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0050] Non-naturally occurring variants of the enzyme peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0051] Variant enzyme peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0052] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0053] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as enzyme activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0054] The present invention further provides fragments of the enzymepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0055] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a enzyme peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the enzyme peptide or could be chosen forthe ability to perform a fiction, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe enzyme peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0056] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0057] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0058] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2^(nd)Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62(1992)).

[0059] Accordingly, the enzyme peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature enzyme peptide is fused withanother compound, such as a compound to increase the half-life of theenzyme peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature enzyme peptide or a pro-protein sequence.

[0060] Protein/Peptide Uses

[0061] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0062] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0063] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. Experimental data as providedin FIG. 1 indicates that the enzymes of the present invention areexpressed in humans in the lung, amygdala, adrenal gland, and fetus, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in the hippocampus. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of enzyme proteins, particularly members of themetalloprotease subfamily (see Background of the Invention). Thestructural and functional information provided in the Background andFigures provide specific and substantial uses for the molecules of thepresent invention, particularly in combination with the expressioninformation provided in FIG. 1. Experimental data as provided in FIG. 1indicates expression in humans in the lung, amygdala, adrenal gland,hippocampus, and fetus. Such uses can readily be determined using theinformation provided herein, that which is known in the art, and routineexperimentation.

[0064] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to enzymes that are related tomembers of the metalloprotease subfamily. Such assays involve any of theknown enzyme functions or activities or properties useful for diagnosisand treatment of enzyme-related conditions that are specific for thesubfamily of enzymes that the one of the present invention belongs to,particularly in cells and tissues that express the enzyme. Experimentaldata as provided in FIG. 1 indicates that the enzymes of the presentinvention are expressed in humans in the lung, amygdala, adrenal gland,and fetus, as indicated by virtual northern blot analysis. In addition,PCR-based tissue screening panels indicate expression in thehippocampus.

[0065] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the enzyme, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the lung, amygdala, adrenal gland,hippocampus, and fetus. In an alternate embodiment, cell-based assaysinvolve recombinant host cells expressing the enzyme protein.

[0066] The polypeptides can be used to identify compounds that modulateenzyme activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the enzyme.Both the enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0067] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the enzyme protein and a molecule that normally interacts withthe enzyme protein, e.g. a substrate or a component of the signalpathway that the enzyme protein normally interacts (for example, anotherenzyme). Such assays typically include the steps of combining the enzymeprotein with a candidate compound under conditions that allow the enzymeprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the enzymeprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0068] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0069] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0070] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

[0071] Any of the biological or biochemical functions mediated by theenzyme can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the enzyme can be assayed.Experimental data as provided in FIG. 1 indicates that the enzymes ofthe present invention are expressed in humans in the lung, amygdala,adrenal gland, and fetus, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in the hippocampus.

[0072] Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

[0073] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the enzyme (e.g. binding partners and/or ligands).Thus, a compound is exposed to a enzyme polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble enzyme polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble enzyme polypeptide, itdecreases the amount of complex formed or activity from the enzymetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the enzyme.Thus, the soluble polypeptide that competes with the target enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0074] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the enzyme protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0075] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein canbe,provided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a enzyme-binding protein and a candidate compound are incubated inthe enzyme protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theenzyme protein target molecule, or which are reactive with enzymeprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0076] Agents that modulate one of the enzymes of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0077] Modulators of enzyme protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the enzyme pathway, by treating cells or tissuesthat express the enzyme. Experimental data as provided in FIG. 1indicates expression in humans in the lung, amygdala, adrenal gland,hippocampus, and fetus. These methods of treatment include the steps ofadministering a modulator of enzyme activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

[0078] In yet another aspect of the invention, the enzyme proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

[0079] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

[0080] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0081] The enzyme proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the lung, amygdala, adrenal gland,hippocampus, and fetus. The method involves contacting a biologicalsample with a compound capable of interacting with the enzyme proteinsuch that the interaction can be detected. Such an assay can be providedin a single detection format or a multi-detection format such as anantibody chip array.

[0082] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0083] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0084] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0085] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0086] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the lung, amygdala, adrenal gland, hippocampus,and fetus. Accordingly, methods for treatment include the use of theenzyme protein or fragments.

[0087] Antibodies

[0088] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0089] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0090] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0091] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0092] Antibodies are preferably prepared from regions or discretefragments of the enzyme proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or enzyme/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0093] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0094] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0095] Antibody Uses

[0096] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in thelung, amygdala, adrenal gland, and fetus, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in the hippocampus. Further, such antibodies can beused to detect protein in situ, in vitro, or in a cell lysate orsupernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0097] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in the lung, amygdala, adrenal gland,hippocampus, and fetus. If a disorder is characterized by a specificmutation in the protein, antibodies specific for this mutant protein canbe used to assay for the presence of the specific mutant protein.

[0098] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the lung, amygdala, adrenal gland, hippocampus, and fetus. Thediagnostic uses can be applied, not only in genetic testing, but also inmonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

[0099] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0100] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in the lung,amygdala, adrenal gland, hippocampus, and fetus. Thus, where a specificprotein has been correlated with expression in a specific tissue,antibodies that are specific for this protein can be used to identify atissue type.

[0101] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the enzyme peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0102] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0103] Nucleic Acid Molecules

[0104] The present invention further provides isolated nucleic acidmolecules that encode a enzyme peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the enzyme peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0105] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0106] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0107] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0108] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0109] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0110] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO: 3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO: 2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0111] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0112] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0113] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the enzyme peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0114] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0115] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon- conservative amino acid substitutions.

[0116] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments-include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0117] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0118] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0119] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel enzyme of the present invention is located on a genome componentthat has been mapped to human chromosome 3 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data.

[0120]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme of the present invention. SNPs were identifiedat 4 different nucleotide positions. Some of these SNPs that are locatedoutside the ORF and in introns may affect gene transcription.

[0121] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2× SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0122] Nucleic Acid Molecule Uses

[0123] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 4 different nucleotidepositions.

[0124] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0125] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0126] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0127] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0128] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the novelenzyme of the present invention is located on a genome component thathas been mapped to human chromosome 3 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.

[0129] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0130] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0131] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0132] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0133] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0134] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the enzymes of the present invention are expressed inhumans in the lung, amygdala, adrenal gland, and fetus, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in the hippocampus. Accordingly, the probescan be used to detect the presence of, or to determine levels of, aspecific nucleic acid molecule in cells, tissues, and in organisms. Thenucleic acid whose level is determined can be DNA or RNA. Accordingly,probes corresponding to the peptides described herein can be used toassess expression and/or gene copy number in a given cell, tissue, ororganism. These uses are relevant for diagnosis of disorders involvingan increase or decrease in enzyme protein expression relative to normalresults.

[0135] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0136] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a enzyme protein, such as bymeasuring a level of a enzyme-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a enzymegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in thelung, amygdala, adrenal gland, and fetus, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in the hippocampus.

[0137] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate enzyme nucleic acid expression.

[0138] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the enzyme gene, particularly biological and pathologicalprocesses that are mediated by the enzyme in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in the lung, amygdala, adrenal gland, hippocampus, and fetus.The method typically includes assaying the ability of the compound tomodulate the expression of the enzyme nucleic acid and thus identifyinga compound that can be used to treat a disorder characterized byundesired enzyme nucleic acid expression. The assays can be performed incell-based and cell-free systems. Cell-based assays include cellsnaturally expressing the enzyme nucleic acid or recombinant cellsgenetically engineered to express specific nucleic acid sequences.

[0139] The assay for enzyme nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the enzyme proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0140] Thus, modulators of enzyme gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of enzyme mRNA inthe presence of the candidate compound is compared to the level ofexpression of enzyme mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0141] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate enzyme nucleic acid expressionin cells and tissues that express the enzyme. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the lung, amygdala, adrenal gland, and fetus,as indicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in the hippocampus.Modulation includes both up-regulation (i.e. activation or agonization)or down-regulation (suppression or antagonization) or nucleic acidexpression.

[0142] Alternatively, a modulator for enzyme nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theenzyme nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in the lung, amygdala, adrenal gland, hippocampus, and fetus.

[0143] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound.

[0144] Accordingly, such monitoring would allow either increasedadministration of the compound or the administration of alternativecompounds to which the patient has not become resistant. Similarly, ifthe level of nucleic acid expression falls below a desirable level,administration of the compound could be commensurately decreased.

[0145] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in enzyme nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

[0146] Individuals carrying mutations in the enzyme gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeof the present invention. SNPs were identified at 4 different nucleotidepositions. Some of these SNPs that are located outside the ORF and inintrons may affect gene transcription. The gene encoding the novelenzyme of the present invention is located on a genome component thathas been mapped to human chromosome 3 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.Genomic DNA can be analyzed directly or can be amplified by using PCRprior to analysis. RNA or cDNA can be used in the same way. In someuses, detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0147] Alternatively, mutations in a enzyme gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0148] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0149] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant enzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0150] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0151] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the enzyme gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeof the present invention. SNPs were identified at 4 different nucleotidepositions. Some of these SNPs that are located outside the ORF and inintrons may affect gene transcription.

[0152] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0153] The nucleic acid molecules are thus useful as antisenseconstructs to control enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of enzyme protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into enzyme protein.

[0154] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

[0155] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in enzyme geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired enzymeprotein to treat the individual.

[0156] The invention also encompasses kits for detecting the presence ofa enzyme nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the lung, amygdala, adrenal gland, and fetus,as indicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in the hippocampus. Forexample, the kit can comprise reagents such as a labeled or labelablenucleic acid or agent capable of detecting enzyme nucleic acid in abiological sample; means for determining the amount of enzyme nucleicacid in the sample; and means for comparing the amount of enzyme nucleicacid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect enzyme protein mRNA or DNA.

[0157] Nucleic Acid Arrays

[0158] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0159] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0160] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0161] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0162] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationW095/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0163] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0164] Using such arrays, the present invention provides methods toidentify the expression of the enzyme proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the enzymegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the enzyme of the presentinvention. SNPs were identified at 4 different nucleotide positions.Some of these SNPs that are located outside the ORF and in introns mayaffect gene transcription.

[0165] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0166] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0167] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0168] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0169] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0170] Vectors/Host Cells

[0171] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0172] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0173] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0174] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0175] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.Coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0176] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0177] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0178] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0179] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0180] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0181] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0182] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroenzyme. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al, Gene 69:301-315 (1988)) and pET 11d (Studier et al, GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0183] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0184] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSecl (Baldari, et al, EMBO J.6:229-234 (1987)), pMFa (Kujan et al, Cell 30:933-943(1982)), pJRY88(Schultz et al, Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calf.).

[0185] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al, Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al, Virology 170:31-39 (1989)).

[0186] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0187] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0188] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0189] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0190] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0191] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0192] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0193] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0194] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell- free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0195] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asenzymes, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0196] Where the peptide is not secreted into the medium, which istypically the case with enzymes, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0197] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0198] Uses of Vectors and Host Cells

[0199] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga enzyme protein or peptide that can be further purified to producedesired amounts of enzyme protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0200] Host cells are also useful for conducting cell-based assaysinvolving the enzyme protein or enzyme protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native enzyme protein is useful forassaying compounds that stimulate or inhibit enzyme protein function.

[0201] Host cells are also useful for identifying enzyme protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantenzyme protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native enzyme protein.

[0202] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0203] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0204] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence., This includesintronic sequences and polyadenylation signals, if not already included.A tissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

[0205] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0206] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0207] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos.WO97/07668 and WO97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growth,cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0208] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfunctions.

[0209] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 3377 DNA Human 1 tcgcggcggc cgtgatggct ggtgacggcg gggccgggcaggggaccggg gccgcggccc 60 gggagcgggc cagctgccgg gagccctgaa tcaccgcctggcccgactcc accatgaacg 120 tcgcgctgca ggagctggga gctggcagca acatggtggagtacaaacgg gccacgcttc 180 gggatgaaga cgcacccgag acccccgtag agggcggggcctccccggac gccatggagg 240 tgggcaaggg ggcttcccct ttctcaccag gccccagccctggcatgacg cctggcacac 300 ccaggagctc tgggctgttc tggagggtca cctgcccccacctccgctcc atctctggcc 360 tctgctctag gactatggtg ggattccaga aggggacaagacagctgtta ggctcacgca 420 cgcagctgga gctggtctta gcaggtgcct ctctactgctggctgcactg cttctgggct 480 gccttgtggc cctaggggtc cagtaccaca gagacccatcccacagcacc tgccttacag 540 aggcctgcat tcgagtggct ggaaaaatcc tggagtccctggaccgaggg gtgagcccct 600 gtgaggactt ttaccagttc tcctgtgggg gctggattcggaggaacccc ctgcccgatg 660 ggcgttctcg ctggaacacc ttcaacagcc tctgggaccaaaaccaggcc atactgaagc 720 acctgcttga aaacaccacc ttcaactcca gcagtgaagctgagcagaag acacagcgct 780 tctacctatc ttgcctacag gtggagcgca ttgaggagctgggagcccag ccactgagag 840 acctcattga gaagattggt ggttggaaca ttacggggccctgggaccag gacaacttta 900 tggaggtgtt gaaggcagta gcagggacct acagggccaccccattcttc accgtctaca 960 tcagtgccga ctctaagagt tccaacagca atgttatccaggtggaccag tctgggctct 1020 ttctgccctc tcgggattac tacttaaaca gaactgccaatgagaaagtg ctcactgcct 1080 atctggatta catggaggaa ctggggatgc tgctgggtgggcggcccacc tccacgaggg 1140 agcagatgca gcaggtgctg gagttggaga tacagctggccaacatcaca gtgccccagg 1200 accagcggcg cgacgaggag aagatctacc acaagatgagcatttcggag ctgcaggctc 1260 tggcgccctc catggactgg cttgagttcc tgtctttcttgctgtcacca ttggagttga 1320 gtgactctga gcctgtggtg gtgtatggga tggattatttgcagcaggtg tcagagctca 1380 tcaaccgcac ggaaccaagc atcctgaaca attacctgatctggaacctg gtgcaaaaga 1440 caacctcaag cctggaccga cgctttgagt ctgcacaagagaagctgctg gagaccctct 1500 atggcactaa gaagtcctgt gtgccgaggt ggcagacctgcatctccaac acggatgacg 1560 cccttggctt tgctttgggg tccctcttcg tgaaggccacgtttgaccgg caaagcaaag 1620 aaattgcaga ggggatgatc agcgaaatcc ggaccgcatttgaggaggcc ctgggacagc 1680 tggtttggat ggatgagaag acccgccagg cagccaaggagaaagcagat gccatctatg 1740 atatgattgg tttcccagac tttatcctgg agcccaaagagctggatgat gtttatgacg 1800 ggtacgaaat ttctgaagat tctttcttcc aaaacatgttgaatttgtac aacttctctg 1860 ccaaggttat ggctgaccag ctccgcaagc ctcccagccgagaccagtgg agcatgaccc 1920 cccagacagt gaatgcctac taccttccaa ctaagaatgagatcgtcttc cccgctggca 1980 tcctgcaggc ccccttctat gcccgcaacc accccaaggccctgaacttc ggtggcatcg 2040 gtgtggtcat gggccatgag ttgacgcatg cctttgatgaccaagggcgc gagtatgaca 2100 aagaagggaa cctgcggccc tggtggcaga atgagtccctggcagccttc cggaaccaca 2160 cggcctgcat ggaggaacag tacaatcaat accaggtcaatggggagagg ctcaacggcc 2220 gccagacgct gggggagaac attgctgaca acggggggctgaaggctgcc tacaatgctt 2280 acaaagcatg gctgagaaag catggggagg agcagcaactgccagccgtg gggctcacca 2340 accaccagct cttcttcgtg ggatttgccc aggtgtggtgctcggtccgc acaccagaga 2400 gctctcacga ggggctggtg accgaccccc acagccctgcccgcttccgc gtgctgggca 2460 ctctctccaa ctcccgtgac ttcctgcggc acttcggctgccctgtcggc tcccccatga 2520 acccagggca gctgtgtgag gtgtggtaga cctggatcaggggagaaatg cccagctgtc 2580 accagacctg gggcagctct cctgacaaag ctgtttgctcttgggttggg aggaagcaaa 2640 tgcaagctgg gctgggtcta gtccctcccc cccacaggtgacatgagtac agaccctcct 2700 caatcaccac attgtgcctc tgctttgggg gtgcccctgcctccagcaga gcccccacca 2760 ttcactgtga catctttccg tgtcaccctg cctggaagaggtctgggtgg ggaggccagt 2820 tcccatagga aggagtctgc ctcttctgtc cccaggctcactcagcctgg cggccatggg 2880 gcctgccgtg cctgccccac tgtgacccac aggcctgggtggtgtacctc ctggacttct 2940 ccccaggctc actcagtgcg cacttagggg tggactcagctctgtctggc tcaccctcac 3000 gggctacccc cacctcaccc tgtgctcctt gtgccactgctcccagtgct gctgctgacc 3060 ttcactgaca gctcctagtg gaagcccaag ggcctctgaaagcctcctgc tgcccactgt 3120 ttccctgggc tgagagggga agtgcatatg tgtagcgggtactggttcct gtgtcttagg 3180 gcacaagcct tagcaaatga ttgattctcc ctggacaaagcaggaaagca gatagagcag 3240 ggaaaaggaa gaacagagtt tatttttaca gaaaagagggtgggagggtg tggtcttggc 3300 ccttatagga ccctgtgcca ataaacagac atgcatccgtcaaaaaaaaa aaaaaaaaaa 3360 aaaaaaaaaa aaaaaaa 3377 2 811 PRT Human 2 MetAsn Val Ala Leu Gln Glu Leu Gly Ala Gly Ser Asn Met Val Glu 1 5 10 15Tyr Lys Arg Ala Thr Leu Arg Asp Glu Asp Ala Pro Glu Thr Pro Val 20 25 30Glu Gly Gly Ala Ser Pro Asp Ala Met Glu Val Gly Lys Gly Ala Ser 35 40 45Pro Phe Ser Pro Gly Pro Ser Pro Gly Met Thr Pro Gly Thr Pro Arg 50 55 60Ser Ser Gly Leu Phe Trp Arg Val Thr Cys Pro His Leu Arg Ser Ile 65 70 7580 Ser Gly Leu Cys Ser Arg Thr Met Val Gly Phe Gln Lys Gly Thr Arg 85 9095 Gln Leu Leu Gly Ser Arg Thr Gln Leu Glu Leu Val Leu Ala Gly Ala 100105 110 Ser Leu Leu Leu Ala Ala Leu Leu Leu Gly Cys Leu Val Ala Leu Gly115 120 125 Val Gln Tyr His Arg Asp Pro Ser His Ser Thr Cys Leu Thr GluAla 130 135 140 Cys Ile Arg Val Ala Gly Lys Ile Leu Glu Ser Leu Asp ArgGly Val 145 150 155 160 Ser Pro Cys Glu Asp Phe Tyr Gln Phe Ser Cys GlyGly Trp Ile Arg 165 170 175 Arg Asn Pro Leu Pro Asp Gly Arg Ser Arg TrpAsn Thr Phe Asn Ser 180 185 190 Leu Trp Asp Gln Asn Gln Ala Ile Leu LysHis Leu Leu Glu Asn Thr 195 200 205 Thr Phe Asn Ser Ser Ser Glu Ala GluGln Lys Thr Gln Arg Phe Tyr 210 215 220 Leu Ser Cys Leu Gln Val Glu ArgIle Glu Glu Leu Gly Ala Gln Pro 225 230 235 240 Leu Arg Asp Leu Ile GluLys Ile Gly Gly Trp Asn Ile Thr Gly Pro 245 250 255 Trp Asp Gln Asp AsnPhe Met Glu Val Leu Lys Ala Val Ala Gly Thr 260 265 270 Tyr Arg Ala ThrPro Phe Phe Thr Val Tyr Ile Ser Ala Asp Ser Lys 275 280 285 Ser Ser AsnSer Asn Val Ile Gln Val Asp Gln Ser Gly Leu Phe Leu 290 295 300 Pro SerArg Asp Tyr Tyr Leu Asn Arg Thr Ala Asn Glu Lys Val Leu 305 310 315 320Thr Ala Tyr Leu Asp Tyr Met Glu Glu Leu Gly Met Leu Leu Gly Gly 325 330335 Arg Pro Thr Ser Thr Arg Glu Gln Met Gln Gln Val Leu Glu Leu Glu 340345 350 Ile Gln Leu Ala Asn Ile Thr Val Pro Gln Asp Gln Arg Arg Asp Glu355 360 365 Glu Lys Ile Tyr His Lys Met Ser Ile Ser Glu Leu Gln Ala LeuAla 370 375 380 Pro Ser Met Asp Trp Leu Glu Phe Leu Ser Phe Leu Leu SerPro Leu 385 390 395 400 Glu Leu Ser Asp Ser Glu Pro Val Val Val Tyr GlyMet Asp Tyr Leu 405 410 415 Gln Gln Val Ser Glu Leu Ile Asn Arg Thr GluPro Ser Ile Leu Asn 420 425 430 Asn Tyr Leu Ile Trp Asn Leu Val Gln LysThr Thr Ser Ser Leu Asp 435 440 445 Arg Arg Phe Glu Ser Ala Gln Glu LysLeu Leu Glu Thr Leu Tyr Gly 450 455 460 Thr Lys Lys Ser Cys Val Pro ArgTrp Gln Thr Cys Ile Ser Asn Thr 465 470 475 480 Asp Asp Ala Leu Gly PheAla Leu Gly Ser Leu Phe Val Lys Ala Thr 485 490 495 Phe Asp Arg Gln SerLys Glu Ile Ala Glu Gly Met Ile Ser Glu Ile 500 505 510 Arg Thr Ala PheGlu Glu Ala Leu Gly Gln Leu Val Trp Met Asp Glu 515 520 525 Lys Thr ArgGln Ala Ala Lys Glu Lys Ala Asp Ala Ile Tyr Asp Met 530 535 540 Ile GlyPhe Pro Asp Phe Ile Leu Glu Pro Lys Glu Leu Asp Asp Val 545 550 555 560Tyr Asp Gly Tyr Glu Ile Ser Glu Asp Ser Phe Phe Gln Asn Met Leu 565 570575 Asn Leu Tyr Asn Phe Ser Ala Lys Val Met Ala Asp Gln Leu Arg Lys 580585 590 Pro Pro Ser Arg Asp Gln Trp Ser Met Thr Pro Gln Thr Val Asn Ala595 600 605 Tyr Tyr Leu Pro Thr Lys Asn Glu Ile Val Phe Pro Ala Gly IleLeu 610 615 620 Gln Ala Pro Phe Tyr Ala Arg Asn His Pro Lys Ala Leu AsnPhe Gly 625 630 635 640 Gly Ile Gly Val Val Met Gly His Glu Leu Thr HisAla Phe Asp Asp 645 650 655 Gln Gly Arg Glu Tyr Asp Lys Glu Gly Asn LeuArg Pro Trp Trp Gln 660 665 670 Asn Glu Ser Leu Ala Ala Phe Arg Asn HisThr Ala Cys Met Glu Glu 675 680 685 Gln Tyr Asn Gln Tyr Gln Val Asn GlyGlu Arg Leu Asn Gly Arg Gln 690 695 700 Thr Leu Gly Glu Asn Ile Ala AspAsn Gly Gly Leu Lys Ala Ala Tyr 705 710 715 720 Asn Ala Tyr Lys Ala TrpLeu Arg Lys His Gly Glu Glu Gln Gln Leu 725 730 735 Pro Ala Val Gly LeuThr Asn His Gln Leu Phe Phe Val Gly Phe Ala 740 745 750 Gln Val Trp CysSer Val Arg Thr Pro Glu Ser Ser His Glu Gly Leu 755 760 765 Val Thr AspPro His Ser Pro Ala Arg Phe Arg Val Leu Gly Thr Leu 770 775 780 Ser AsnSer Arg Asp Phe Leu Arg His Phe Gly Cys Pro Val Gly Ser 785 790 795 800Pro Met Asn Pro Gly Gln Leu Cys Glu Val Trp 805 810 3 19650 DNA Humanmisc_feature (1)...(19650) n = A,T,C or G 3 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnncacc ttagacttgacaggcctgct tagtcggact ctaaagcacc cctttgcttt 1080 tcgttaaata ttgcttggtgttagtttttt ttctccttgt aaatctccca aataaaacgg 1140 tttgctttcc ccaagttagaagtgttagca cgtcttttct ttaaatatct gtgcatggct 1200 gtttttttcc ctgccaatttgtcaccatct gtaaccctcc ctttatgaga cgatctgatg 1260 acagcagtta tcttggagagtagaagtgtg gtcttgaagc gccatggaag agtagagtca 1320 gtgtatgctg tgtgtgtgtggagtgtatgc tccccctgca cttggtgtgt gtacatacag 1380 aaacacagtg tgcgtgtgtgttggctctgg gtgtgttgtg cgtgtgtaca ctgtgtgtga 1440 gtatgcagtg tgtgtacattctgtgggcat ctcgtgtgtg tgtggactgt gtgctgggcg 1500 tcgtgcctgc ccgtgtccttggcgccttgg cgtctatgcg ttctctgcac ataggtaggt 1560 accacgtgca caccctgaatgtgagtgaac tgcctgtgtg ctatgtattt gccggctgaa 1620 gaggggctgt gtggactactgggggaagac gttcctcang agggcataat ttctctaaag 1680 tgcttaaagg ggatggagagagcctgaaat ttgggggaag taggccaagg agtattatca 1740 acgtctgggc ctggttgaatttcattactt ttcctaggaa agtaaattat gggtggcttg 1800 aaggagggtg ctgctgagatggggggcgga ccatgaagcg tggaggggtc tccggtgttg 1860 ctggagggca gctggagcctgcggagagcc tcggcgcgct cctccctctc ccccaccctc 1920 cccccacccc gggcggggctccgcgtgggg cggtggactc gggcgggggg gggggcggcc 1980 gcggccgagc gggggtgctgcgcggcggcc gtgatggctg gtgacggcgg ggccgggcag 2040 gggaccgggg ccgcggcccgggagcgggcc agctgccggg agccctgaat caccgcctgg 2100 cccgactcca ccatgaacgtcgcgctgcag gagctgggag ctggcagcaa cgtgagtggg 2160 ggccccgggc tccacgggaggggactgggt ggagggggac gaggcagagg ggtcggccgc 2220 ggaggggcag gcggtgcccggctcgcggag gtaaggctgc ctcccgggcc tggtggaggg 2280 gtgatagaga gaccccgggcccgagagcag ggcaggtggg aagggaaggg ccctcttagc 2340 agggcggagg ggtccgcgaggcagggagca ctggggcagg gtcgtgggca aatagccctc 2400 tctgcctgac ctcggttggcaaccccgact gtctggcaga tggtggagta caaacgggcc 2460 acgcttcggg atgaagacgcacccgagacc cccgtagagg gcggggcctc cccggacgcc 2520 atggaggtgg gcaagggggcttcccctttc tcaccaggcc ccagccctgg catgacgcct 2580 ggcacaccca ggagctctgggctgttctgg agggtcatct gcccccacct ccgctccatc 2640 tctggcctct gctctaggactatggtgagg cgatgctaag ccgtgacgtt gcacaaaaca 2700 gactcaaggc tcaactcactggctggcctc attgcccccg ggcccagagt taaccctgtg 2760 gctctgaaaa ctgcctgtggcttcaccctc tggtaatctt ggatccctgc cctgcatctc 2820 agtcactctc tgtccccctgtgttccccag gtgggattcc agaaggggac aagacagctg 2880 ttaggctcac gcacgcagctggagctggtc ttagcaggtg cctctctact gctggctgca 2940 ctgcttctgg gctgccttgtggccctaggg gtccagtacc acagaggtag gtgggcccac 3000 actcttcgtc agtattcataactaggggtt ctggaggcct aagggcctct aagattttca 3060 cttgtgggaa ccaagccttccctgcagaaa agcccccggc tttgctttct cttcccaacc 3120 ttcctgctgt catggcccttgcagagtttg cctcttccag acagacagac tgacagtctc 3180 ctaccctccg gccatgttccctaccacaga cccatcccac agcacctgcc ttacagaggc 3240 ctgcattcga gtggctggaaaaatcctgga gtccctggac cgaggggtga gcccctgtga 3300 ggacttttac cagttctcctgtgnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnc ttagcaaata ggcagtgtcc 3480 catgaatgag gaagtggatggttctgtgaa cactcccaga gggtggggag gcagagagca 3540 ggggactatt gagaagtgcagatgggtttg atgggggcag aactctgggt acaatggagg 3600 gccgcttctc tgcactctgtttggagcact gtcgtggtgt ggtagacacc agggagcctg 3660 tactgcttag atatccttgggtctccatgg acagggagag gaagccacgg cttgctgttt 3720 cagacactct tcctgggtctgcgttagcag gactgctcat tgacaaggca aggagagaaa 3780 ccgagcaagg gccagggactccccctcagc agttaacgta attgccacct ggatcctgtg 3840 ttctgcccca cagaaaacaccaccttcaac tccagcagtg aagctgagca gaagacacag 3900 cgcttctacc tatcttgcctacaggtggag cgcattgagg agctgggagc ccagccactg 3960 agagacctca ttgagaaggtagggccactg agccggttga gggcagggga gcaggagagg 4020 ccttgagaga ggagatggcccaggaacgct ttgggagctc ctgcactaat cattccactt 4080 atggtctcta catagattggtggttggaac attacggggc cctgggacca ggacaacttt 4140 atggaggtgt tgaaggcagtagcagggacc tacagggcca ccccattctt caccgtctac 4200 atcagtgccg actctaagagttccaacagc aatgttatcc aggtgatgag ctgggaaagg 4260 gtggggagag acttagggacactttgctga gcccagactt ccctctcctg tgacaggcag 4320 gctgggctga ccccccggccccacccctac ccccgctcgg gaattcaggt tcccatggtg 4380 gggaaagcga ggggctcacctcctttcctt gacattgcag gtggaccagt ctgggctctt 4440 tctgccctct cgggattactacttaaacag aactgccaat gagaaagtaa ggaacatctt 4500 ccgaaccccc atccctacccctggctgagc tgggctgatc cctgttgact tttccctttg 4560 ccaagggtca gagcagggaaggtgagccta tcctgtcacc tagtgaacaa actgcccctc 4620 ctttctttct tcttttcttcctccctccct ccctttcttc cccttttcct tccttccttc 4680 ctcttattct tctagtaggtttcatagaca cctactgtgt gccaggtcca gtgggggaat 4740 tctgagatat aagtttnccgagcccattgc cagcaggaga ggggatcctt tagagtcgca 4800 caaacaggtc agtcaagtctaaagacnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5100 nnnnnnnnnn nnnnnnnnnngcctgnactt gcatgcaccg cggttcggct nctagnagna 5160 tccccccact gcactccagcctgggtgacg gagagagact ccgactcaaa aaaaaaaaaa 5220 aaaaagaaag aaaaagaaagaaggaacagt ttaaacaaaa gtgttgatga ggctgagcac 5280 agtggctcac acctgtaatccccgcacttt gggaggctga ggccggcgga tcacttgagg 5340 ttaggagttc aagaccaggctggcctacaa ggtgaaaacc cgtctctact aaaaatacaa 5400 aaattagcca ggcatggtggtgtgcacctg taatctcagc tacttgggag gctgaggcaa 5460 agagaatcgc ttgaatccaggaggcagagg ttgcagtgag ctgagatggc accactgcac 5520 tccagcctgg gcaacagaacaagacttcat ctcaaaaaaa aaaaaaaaag tgttgacgag 5580 ggaaaggcta ggtgtgtctggaccatggca aggggtccac tgtggtaaaa tatagaactc 5640 aaggcagatg agaggctggagaggtgggca ggaatgggtt atggagggga ccttgaatag 5700 cacactacgg agtttattctgtagctcccg gagagccatt gcatgctcca aagtagggag 5760 ggagcgcant gctttgggaagtcagtttgt ttggggtgtg aagagtanat gtgagaacnn 5820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnag ttccaggccc acccttgggc 11460 caaacatgtt gaagaccgccatgctgtagc tagaacttac aaaagatgta agcctgggca 11520 taggtggccg ggtgccgttgtggtcgccac gctatcttgg ggagggatta agggcaagga 11580 aaattcacct tgaggcccaaggaaggcaca agggttatca cgtgaagccg aggatcacca 11640 tcaccatgca ctaacacgccttgggcaagc acgaagcgag gagttgccat ctcaaaacaa 11700 aaacgaaaaa caaacaaacaaaatgctaat caactgtcat tggtaaggct tctggtcaac 11760 agtatgctgt caatagttaagtttttgggc tgggcgcagt ggctcacgcc tgtaatccca 11820 gcactttggg aggccaaagcgggtagatca cctgaggtca ggagtcgaga ctagcctggc 11880 caacatggcg aaacccagtctctactaaaa atataaaaat tagccaggcg tggtggtggg 11940 cacttgtaat cccagctactcaggaagctg aggcagaact gcttgaactg ggaagtggag 12000 gttgcagtga gccgagatcgtgccattgca ttccagcctg ggcgacaaga gcaaaactcc 12060 atctcaaaaa aaaaaaaaaaaaaaaaagtt gtttttgggg agtcaaaaat gaggccaggc 12120 gcagtggctc atgcctgtaatcacagcact ttgggaggcc gaggcgggtg gatcacctga 12180 ggtcaggagt tcgtgaccagcttggccaac ctggtgaaac cccgtctcta ctaaaaatac 12240 aaaaattagc cgggcatggtggcgggcgcc cgtaatctca gctacttggg cggctgaggc 12300 aggagaattg cttcaacccgggaggcagag gttgcaatga gctgagatcg cgccactgca 12360 ctccagcctt ggcgacagagggagactcca tgtcaaatta aaaaaaagac cccaggattt 12420 tggactgtgc aggggtcggtgccccaaacc cccacgttgt tcaaggtcaa ctgtacactg 12480 tcatagtcgg gaaaacttcatcactgcagc tgctcctgtt tcttgaaacc tgaagcggga 12540 aactggatcc tgggacactactgcccccta tcgcctgttg gtcttcaaag aaataatccc 12600 ttcaattttg caaggcctgtggtgtcattc ccttttaaca gataaggaaa ccgaggccag 12660 gacgtggtgg aaaataatcaaggtcacaca tctatgtgca aaagtggagt aacaacccag 12720 gctcctcatt cccaggtcagtccagtgacc tcaattgaca tgaaatgtgt gaggtccttc 12780 tgtggccctg tggcagggcctgaagaggac agcgtatgta aatcaagtct tgtgccttca 12840 tgagtgaggc agagtagaaaataacagtaa ttcactagga ccgaatctgc attgtaaaca 12900 gagaggaaag ggctagtatttggcagaagg atgtcaagga acattttaga gataagaggt 12960 gacatttggg ttctgagggatgagtaggag tgtgccaggg tgcaaaggat gaaaagacag 13020 ctctagcagc tggtaagggctaaggggcat ggagaaacag caagactttg gggaactggt 13080 agaattctaa ttctggaaaatttgaacaag gtaatttttt gtgtgtggtt aaggtattac 13140 atacatacag taaaataaaatgcaatagtt gctgggtgtg gaggctcacg cctgttaatc 13200 ccagtacttt ggaaggcagaggcgggtgga tcatctgaag gtcaggagtt cgagaccagc 13260 ctgaccaaca tggtgaaaacccgtctctac taaaaataca aaaattacct gggtgtggtg 13320 gcaggcgccc gtaatcccagctacttggga ggctaaggga gaagaatagc ttgaaacccg 13380 gaggtggagg ttgcagtgagctgagattgc actattgcgc tccagcctgg gtgacaagag 13440 tgaaaagctg tctcaaaataaaataaaaat gtaatagtct aattgatttt tttaaaaaat 13500 gtagacatcc acgtatctaccacctaggta aagatactag agattccagc aacctgggag 13560 gatccctcgt gcccctttcaggtctatatg agcctccacc gttccccagt cccctggaag 13620 gagagggggt gggagaggcaacatgaaacc taaaaaccag tgggcttcgc gcctgtaatc 13680 ccagctattg ggttggctgaggcaggagga tcacttgccc aggagttgga ggctgcagtg 13740 agctatgatc gcgccaccgcactccagcct gggcgacaga tcaagacccc atctctaagc 13800 aaacaaacaa ataaacacccctcaaaaccc atggcttcag gcctggcgcg gtagcttact 13860 tctgtaatct cagcactttgggaggccgag gagggcggat cacttgaggt caggagttcc 13920 agaccagact ggccaacatggcgaaacccc gtctctacta aaaaataaaa aaaaaaaaaa 13980 attggccggg cgcggtggctcacacctgta attaccagca gnnnnnnnnn nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nttttaaaga atgtagacat ccacgtatct 14100 accacctagg tagagatactagagattcca gcaacctggg aggatccctc gtgcgccttt 14160 caggtctata tgagcctccaccgttcccca gtcccctgga aggagagggg gtgggagagg 14220 caacatgaaa cctaaaaaccagtgggcttc gcgcctgtaa tcccagctat tgggttggct 14280 gaggcaggag gatcacttgcccaggagttg gaggctgcag tgagctatga tcgcgccacc 14340 gcactccagc ctgggcgacagatcaagacc ccatctctaa gcaaacaaac aaataaacac 14400 ccctcaaaac ccatggcttcaggcctggcg cggtagctta cttctgtaat ctcagcactt 14460 tgggaggtca aggtgggcggatcacttgaa gtaaggagtt caagtaccat cctggctaac 14520 acggtgaaac cccgtctctactgaaaagac aaaaaattta gccgggcgtg gtggcgggcg 14580 cctttagtct cagctactcgggaggctgag gcaggagaat ggcgtgaacc cgggaggtgg 14640 agcttgcagt gagctgagatcgcaccactg cactccagtc tgggtgacag agtgagactc 14700 catctcaaaa aaaaaaaaaaagaagtcaaa gtagtagaaa ctgctgatag actgaatgtg 14760 gggggttagg gagatggaggaagctgagtg actcccaggt ttcttgcatg ggggactgac 14820 tggatataaa attagttgtgggccgggcac ggtggctcat gcctttaatc ccagcacttt 14880 gggaggccaa agcgggcagatcacttgagc tcaggagttc aagaccagcc tgggaaacat 14940 ggtgagaccc cttctgtaagggnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000 nnnnnnnnnn nnntgacctttttttggctc tgntcggtca ctagcangca agttattggg 15060 agtctacaag attctttcacactatgccct caaaattgac tgttcatgta tgtgcagaca 15120 tatagaaaaa caacgggagccaggcgcggt ggctcacgcc ggtaatccca gcactttggg 15180 aggccaaggc gggtgaatcatggggtcagg agttcgagac cagcctggcc aacatggtga 15240 aacctggtct ctactaaaaatacaaaaaat tagccgggcg tggtggcggg tgtctgtaat 15300 cccagctact tgggaggctgaggcaggaga atcacttgaa cccaggaggc ggaggttgca 15360 gtgagccgag atcgcgccagtgcactccag cctgggcgac agagcaaaac tctgtctcaa 15420 aaaaaaaaaa aaaaaaaagaaaagaaaaga aaaacaactg gatgtaaatt gatgaacaaa 15480 tgaagtagtg ctgctttgggcagtgggatt ataagagtcc tttaaagttg tctatgtgtt 15540 tatgtttaac tatataactagaagaaatat ttatttatta ggatatgata atggatgtgc 15600 ttaaagtatt acctgtaaggatgtttatgg tttttatggc aatgttgttt ataatagcag 15660 aaaatgagaa caggttaaatgtccaactat agggtaaagg aaaaataaat tgtggttagg 15720 atgggttgtg aggatccttaaatggctgat atatctttca gcaaaaaaag taggttacaa 15780 aaaatatata ccctatacaacataattcca tattttatat gcatatcagg ggagggaaaa 15840 actctagaag tgggtaatcaaaatgttaaa agaacttatc tatgaatgag tgctttataa 15900 ctggtctgtt cttcaattctcaattttcca aattttctgt gaatgtcctc ttttcataat 15960 cagataaaaa tcattgcactaggctgggcg tggtggttca cgcttgtaat cccagcactt 16020 tgggaggctg aggcgggtggatcacgtggt caggagttca agaccaacct ggccaagatg 16080 gtgaaacccc agctctactaaaaatacaaa aattacccgg gcatgatggc gggagcctgt 16140 aatcctagct acttgggaggctgaggcagg agaatcgctt gaactcggga ggcggaggtt 16200 gcagtgagcc gagattgcgccactgcactc catcctaggt aacacagcca gactctgtct 16260 caaaaaaaaa aaaaaatcattgcactatat taaattataa tataatttga tgaacttatt 16320 gtcaattaaa atgtgtacttaattaagaaa aaagccagcc acaatcccag tacctttaca 16380 aatggtgttt ccttctcatcgtctccaggt gctcagccgt atttctttag tctagacgtt 16440 cccatttccc ctgggtggacagggatgggg caccaagggt ggatgggtgg ggcagggatg 16500 cattcagtgc aggggaaggctgactttacc tcctccctcc caggcagagg ggatgatcag 16560 cgaaatccgg accgcatttgaggaggccct gggacagctg gtttggatgg atgagaagac 16620 ccgccaggca gccaaggagaaagtgagcgg tggctagggt tggggcgcca tcttgaggtg 16680 gggttcaagg atacagttttgctaggaacc tggggaagga aacaaaccct taacctggtc 16740 tcttcaggca gatgccatctatgatatgat tggtttccca gactttatcc tggagcccaa 16800 agagctggat gatgtttatgacggggtgag tacctacgct catcagtact gaacttcagc 16860 cctgtagagg gcactgttccctgggcttag aaattggggc tcaagcactg ggaaagaggt 16920 gcttgtcggt ttcttttagaggcagatgga ggtaaccagc attgttaaaa tgttggctct 16980 gtgacaggct gcaggccaaacagcagtgaa atatagtgct aacgagccaa gatttggagt 17040 caagcctaat caaattctgtttctacctct aactttgtaa ccttaacaaa atctctctag 17100 gccttggttt cattttctgtaaaatggggg tcctactagt gccttcctca tagggttgtt 17160 gtgagataaa tgaatacagtatgtaaaaaa acagcaccca taacataaat ggcctttaaa 17220 tattgccaat tatggtttactagatatttt acagttgagg aaactgaggt ttggagagat 17280 actaatgagt agccaaactggcgctattat cttctccaat ggattctctt gctctctgtc 17340 tacttcccaa cttaccacagaacaaannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnagaat caccaacagc 18840 attggatgaa aataaagaagaacaagaggt tcgtttgaga ggaagccggg aaaattctct 18900 cgataaagaa atgcaagtgcgcgcgcggcg caaccactac aatagtgtgt cgtccacccc 18960 agagagtgaa gggggccccccccgccccaa aggaaagggg tagtgtccac gccgctccac 19020 aaagagagag aaggaaagaagtagttttcc cccccccggg gagaaacctt ggatggggct 19080 canccccccc tctttttttttcccgcgaaa acccccccca aaaagttttt tttaaaaaac 19140 aaaaaagggg ggtttggttttttgggcccc gtggcccctt tggtttaaat tgggagaaag 19200 agggcttaaa ggggggattcaagaaaaaac ccccccccaa ttgccccaaa ttgtaatttc 19260 ctaaccccaa aaggggcccctaaaatttcc ggggaaaccc gtgtgggcaa tggcccatta 19320 gtttacccaa tgcctttattgacaaaggta gggccccatg gagtcgtccc ctctagccta 19380 gaattcccag tggctcctgcaagggccttg ggacattgat gtagccccaa gggccctgaa 19440 gtctgtggac cagggctggtggggcactgc tgcccccaag agacgagctc tggttttggt 19500 ggggtgcaaa ggtgagttctcctcagggcg cgagtatgac aaagaaggga actgcggccc 19560 tggtggcaga atgagtccctggcagccttc cggaaccaca cggcctgcat ggaggaacag 19620 tacaatcaat accaggtcaatggggagagg 19650 4 765 PRT Human 4 Met Asn Val Ala Leu Gln Glu Leu GlyAla Gly Ser Asn Met Val Glu 1 5 10 15 Tyr Lys Arg Ala Thr Leu Arg AspGlu Asp Ala Pro Glu Thr Pro Val 20 25 30 Glu Gly Gly Ala Ser Pro Asp AlaMet Glu Val Gly Phe Gln Lys Gly 35 40 45 Thr Arg Gln Leu Leu Gly Ser ArgThr Gln Leu Glu Leu Val Leu Ala 50 55 60 Gly Ala Ser Leu Leu Leu Ala AlaLeu Leu Leu Gly Cys Leu Val Ala 65 70 75 80 Leu Gly Val Gln Tyr His ArgAsp Pro Ser His Ser Thr Cys Leu Thr 85 90 95 Glu Ala Cys Ile Arg Val AlaGly Lys Ile Leu Glu Ser Leu Asp Arg 100 105 110 Gly Val Ser Pro Cys GluAsp Phe Tyr Gln Phe Ser Cys Gly Gly Trp 115 120 125 Ile Arg Arg Asn ProLeu Pro Asp Gly Arg Ser Arg Trp Asn Thr Phe 130 135 140 Asn Ser Leu TrpAsp Gln Asn Gln Ala Ile Leu Lys His Leu Leu Glu 145 150 155 160 Asn ThrThr Phe Asn Ser Ser Ser Glu Ala Glu Gln Lys Thr Gln Arg 165 170 175 PheTyr Leu Ser Cys Leu Gln Val Glu Arg Ile Glu Glu Leu Gly Ala 180 185 190Gln Pro Leu Arg Asp Leu Ile Glu Lys Ile Gly Gly Trp Asn Ile Thr 195 200205 Gly Pro Trp Asp Gln Asp Asn Phe Met Glu Val Leu Lys Ala Val Ala 210215 220 Gly Thr Tyr Arg Ala Thr Pro Phe Phe Thr Val Tyr Ile Ser Ala Asp225 230 235 240 Ser Lys Ser Ser Asn Ser Asn Val Ile Gln Val Asp Gln SerGly Leu 245 250 255 Phe Leu Pro Ser Arg Asp Tyr Tyr Leu Asn Arg Thr AlaAsn Glu Lys 260 265 270 Val Leu Thr Ala Tyr Leu Asp Tyr Met Glu Glu LeuGly Met Leu Leu 275 280 285 Gly Gly Arg Pro Thr Ser Thr Arg Glu Gln MetGln Gln Val Leu Glu 290 295 300 Leu Glu Ile Gln Leu Ala Asn Ile Thr ValPro Gln Asp Gln Arg Arg 305 310 315 320 Asp Glu Glu Lys Ile Tyr His LysMet Ser Ile Ser Glu Leu Gln Ala 325 330 335 Leu Ala Pro Ser Met Asp TrpLeu Glu Phe Leu Ser Phe Leu Leu Ser 340 345 350 Pro Leu Glu Leu Ser AspSer Glu Pro Val Val Val Tyr Gly Met Asp 355 360 365 Tyr Leu Gln Gln ValSer Glu Leu Ile Asn Arg Thr Glu Pro Ser Ile 370 375 380 Leu Asn Asn TyrLeu Ile Trp Asn Leu Val Gln Lys Thr Thr Ser Ser 385 390 395 400 Leu AspArg Arg Phe Glu Ser Ala Gln Glu Lys Leu Leu Glu Thr Leu 405 410 415 TyrGly Thr Lys Lys Ser Cys Val Pro Arg Trp Gln Thr Cys Ile Ser 420 425 430Asn Thr Asp Asp Ala Leu Gly Phe Ala Leu Gly Ser Leu Phe Val Lys 435 440445 Ala Thr Phe Asp Arg Gln Ser Lys Glu Ile Ala Glu Gly Met Ile Ser 450455 460 Glu Ile Arg Thr Ala Phe Glu Glu Ala Leu Gly Gln Leu Val Trp Met465 470 475 480 Asp Glu Lys Thr Arg Gln Ala Ala Lys Glu Lys Ala Asp AlaIle Tyr 485 490 495 Asp Met Ile Gly Phe Pro Asp Phe Ile Leu Glu Pro LysGlu Leu Asp 500 505 510 Asp Val Tyr Asp Gly Tyr Glu Ile Ser Glu Asp SerPhe Phe Gln Asn 515 520 525 Met Leu Asn Leu Tyr Asn Phe Ser Ala Lys ValMet Ala Asp Gln Leu 530 535 540 Arg Lys Pro Pro Ser Arg Asp Gln Trp SerMet Thr Pro Gln Thr Val 545 550 555 560 Asn Ala Tyr Tyr Leu Pro Thr LysAsn Glu Ile Val Phe Pro Ala Gly 565 570 575 Ile Leu Gln Ala Pro Phe TyrAla Arg Asn His Pro Lys Ala Leu Asn 580 585 590 Phe Gly Gly Ile Gly ValVal Met Gly His Glu Leu Thr His Ala Phe 595 600 605 Asp Asp Gln Gly ArgGlu Tyr Asp Lys Glu Gly Asn Leu Arg Pro Trp 610 615 620 Trp Gln Asn GluSer Leu Ala Ala Phe Arg Asn His Thr Ala Cys Met 625 630 635 640 Glu GluGln Tyr Asn Gln Tyr Gln Val Asn Gly Glu Arg Leu Asn Gly 645 650 655 ArgGln Thr Leu Gly Glu Asn Ile Ala Asp Asn Gly Gly Leu Lys Ala 660 665 670Ala Tyr Asn Ala Tyr Lys Ala Trp Leu Arg Lys His Gly Glu Glu Gln 675 680685 Gln Leu Pro Ala Val Gly Leu Thr Asn His Gln Leu Phe Phe Val Gly 690695 700 Phe Ala Gln Val Trp Cys Ser Val Arg Thr Pro Glu Ser Ser His Glu705 710 715 720 Gly Leu Val Thr Asp Pro His Ser Pro Ala Arg Phe Arg ValLeu Gly 725 730 735 Thr Leu Ser Asn Ser Arg Asp Phe Leu Arg His Phe GlyCys Pro Val 740 745 750 Gly Ser Pro Met Asn Pro Gly Gln Leu Cys Glu ValTrp 755 760 765

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 2. An isolated peptide comprising an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 3. An isolated antibody that selectivelybinds to a peptide of claim
 2. 4. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes an amino acid sequence shownin SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the fiction or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human enzyme protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human enzyme peptide having an amino acidsequence that shares at least 70% homology with an amino acid sequenceshown in SEQ ID NO:
 2. 21. A peptide according to claim 20 that sharesat least 90 percent homology with an amino acid sequence shown in SEQ IDNO:
 2. 22. An isolated nucleic acid molecule encoding a human enzymepeptide, said nucleic acid molecule sharing at least 80 percent homologywith a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleicacid molecule according to claim 22 that shares at least 90 percenthomology with a nucleic acid molecule shown in SEQ ID NOS: 1 or 3.